The first medicament to be obtained by engineering a living (bacterial) system was insulin, approved by the Food and Drug Administration (FDA) in 1982. Human growth hormone, previously extracted from cadavers, was also rapidly engineered. In 1986 the FDA approved the first recombinant human vaccine, against hepatitis B. The industrial production of medicaments using living systems as bioreactors has become widespread since then, and is now the preferred synthesis method for numerous medicaments, especially due to the relatively low manufacturing cost.
Many human proteins designed for therapeutic (and non-therapeutic) use are produced by bioengineering technology, including erythropoietin (for the treatment of anaemia), interleukin 2 (for the treatment of renal carcinoma), IL-1Ra (IL-1 receptor antagonist), glucagon, interferon, human DNase enzyme, calcitonin and many others.
Said medicaments mainly have a protein or polypeptide nature, and are generally administered parenterally, because oral administration would cause rapid degradation of the active ingredient. However, parenteral administration can cause problems with patient compliance because of the repeated administrations required to ensure therapeutic efficacy. These proteins generally have a very short half-life; for example, the growth hormone hGH (genetically produced) requires daily injections. Consequently, to reduce the number of administrations and increase patient compliance, many experiments have been conducted to develop systems for the release of proteins/medicaments with an increased half-life.
Said release systems must guarantee that the activity of the medicament is maintained, and must therefore ensure that the three-dimensional structure of the protein/medicament remains intact; however, stress situations which can occur during their preparation, such as the presence of organic solvent and/or variations in temperature and pH, can cause deamidation or oxidation of the protein chain, leading to denaturing and loss of therapeutic activity.
For example, the use of PLGA (polylactic/glycolic acid) microspheres is known as a release system for small peptides, with excellent results (Hutchinson F. G., Biochem. Soc. Trans., 1985, 13:520-523); however, its use to formulate a depot in the release of hGH was impossible, because the denaturing of the protein created inflammatory problems.
The continual search for protein/medicament release systems devoid of toxicity but with unchanged efficacy of the active ingredient delivered has led to the use of natural polymers for formulations which (due to their combination with the active ingredient) can present a modification in the kinetics of the medicament with which they are combined.
Hyaluronic acid (HA) is a heteropolysaccharide composed of alternate residues of D-glucuronic acid and N-acetyl-D-glucosamine. It is a natural straight-chain polymer with a molecular weight ranging between 50,000 and 13×106 Da, depending on the source from which it is obtained and the preparation methods used.
It is present in nature in pericellular gels, in the ground substance of the connective tissue of vertebrates (of which it is one of the main components), and in the synovial fluid of the joints, the vitreous humour and the umbilical cord.
HA therefore plays an important biological part in living organisms by providing a mechanical support for the cells of many tissues, such as skin, tendons, muscles and cartilage.
Due to its properties, hyaluronic acid protects the tissues against free radicals, controls inflammatory processes and stimulates angiogenesis, and has proved particularly effective in modulating all the main stages of wound-healing (EP 1196179).
The use of said polysaccharide as a carrier of various kinds of medicaments is known, in simple combination or salified with hyaluronic acid, because its particular properties of biocompatibility, biodegradability, non-immunogenicity, viscosity and hydratability make it particularly suitable as a release system for medicaments and molecules at both topical and systemic level (EP 197718, EP 445255). Said polysaccharide has also been studied as a drug delivery for specific proteins, such as IL-1Ra (U.S. Pat. No. 6,096,728), erythropoietin (Hahn S K. et al., Int J Pharm., 2006, 28, 322:44-51), insulin (Nomura M. et al., J Pharm Pharmacol, 1994, 46:768-770), growth hormone (Kim S J. et al., Journal of Controlled Release, 2005, 104:323-335), interferon and follicle-stimulating hormone (U.S. Pat. No. 8,025,900).
In all these cases, the combination of the medicament with the polysaccharide has led to “slow” release of the protein carried, but not slow enough to reduce the number of administrations of the medicament.
HA is a completely natural polysaccharide which is rapidly degraded by the enzymes present in the body (hyaluronidase), releasing the medicament with which it is combined relatively rapidly. For these reasons, the carboxyl and hydroxyl groups of HA have been chemically modified to give rise to crosslinked derivatives (U.S. Pat. Nos. 4,582,865; 4,713,448; 5,676,964; 4,957,74; 5,827,937) or derivatised with other natural or synthetic polymers, or with molecules of various sizes and physicochemical characteristics (U.S. Pat. No. 4,851,521); however, the preparation processes of said derivatives often prejudice the integrity of the pharmacological agent conveyed.